The ultimate goal of this proposal is to develop a fully-automated approach for analysis of glycopeptides and to apply that approach to the analysis of several different HIV-Env glycoproteins. This work would be accomplished by completing three specific aims: AIM 1: Using MS data as the input, develop an algorithm that rapidly identifies glycopeptide compositions for singly glycosylated glycopeptides. AIM 2: Develop an algorithm that characterizes multiply glycosylated glycopeptides. AIM 3: Use the tools in AIMS 1 and 2 to screen for accessible and genetically conserved points on the HIV Env protein. The bulk of the work, Aims 1 and 2, would be completed by synergistically incorporating expertise in mass spectral (MS) data acquisition and analysis with expertise in software design/development. This work can broadly impact human health research because glycopeptide analysis is an enabling bioanalytical technology that can be used to screen for biomarkers of disease or disease state; it can be used to help verify the safety and consistency of glycoprotein-based pharmaceuticals; and it can be used to analyze glycopeptides on HIV Envelope proteins, a major target for HIV vaccine development. After completion of the automated glycosylation profiling system (Aims 1 and 2) this system would be used to identify the glycopeptide composition on a variety of HIV- Env vaccine candidates generated in collaboration with Dr. Barton F. Haynes at Duke University Medical Center. The method for this approach includes preparing tryptic digests of each of the glycoproteins, conducing HPLC-MS and LC-MS/MS analyses on the digested products, and interpreting the results using the developed glycopeptide analysis software. After completion of this work, the glycopeptide profiles of the Env proteins would provide information about epitope accessibility on Env. This is relevant to human health because identifying epitopes on the protein that are consistently exposed across HIV-1 clades is a first step in designing an HIV vaccine that mimics these epitopes and elicits broadly neutralizing antibodies to HIV-1.